CA2091243C - Recombinant acc synthase - Google Patents

Abstract

Isolation of the cDNA encoding the ACC synthase of zucchini using a novel isolation method permits the recovery of ACC synthase genes from a variety of higher plant sources. The ACC synthases are coded by multigene families, the members of which may be responsible for various plant development characteristics effected by-ethylene. Recombinant production of ACC
synthase and thereby in vitro production of ACC, ethylene and ethanol is also enabled by use of this gene. In addition, control of the processes in plants which are mediated by ACC synthase can be effected using antisense technology or by the use of mutated ACC synthase genes.

Description

WO 92/04456 _ 1 _ PCT/US91 /06453 w RECOMBINANT ACC SYNTHASE
Field of the Invention The invention relates to the plant enzyme ACC syn thase which is essential for the production of ethylene in higher plants. More particularly, the invention concerns recombinant methods and materials for the production of this enzyme and their use in controlling plant develop-ment, and in particular, plant genescence.
Background Art The enzyme ACC synthase is essential to the produc-tion of ethylene in higher plants. It is well known that ethylene is related to various events in plant growth and development including fruit ripening, seed germination, abscission, and leaf and flower senescence. Ethylene production is strictly regulated by the plant and is induced by a variety of external factors, including the application of auxins, wounding, anaerobic conditions, viral infection, elicitor treatment, chilling, drought, and ions such as cadmium and lithium ions. A review of ethylene production and effects in plants may be found, for example, in Abeles, F.B., "Ethylene in Plant Biology"
(1983) Academic Press, New York.
It is also known that the synthesis of ethylene in higher plants includes a rate limiting step which is the conversion of S-adenosyl methionine (AdoMet) to 1-aminocy clopropane-1-carboxylic acid (ACC). This conversion is catalyzed by the enzyme ACC synthase (EC4.4.1.14). This enzyme has been partially purified from several sources by Nakajima, N., et al, Plant Cell Physiol (1986) 27:969-980;
Mehta, A.M., et al., Proc Natl Acad Sci USA (1988) 85:8810-8814; Nakajima, N., et al., Plant Cell Physiol (1988) 29:989-990; Tsai, D.S., et al., Arch Biochem Biophys (1988) 264:632-640; Bleecker, A.B., et al., Proc Natl Acad Sci USA (1986) 83:7755-7759; Pr'ivale. L.S., et al., Arch Biochem Biophys (1987) 253:333-340; Sato, S., et al., Plant Physiol (1988) 88:109-114; Van Der Straeten, D., et al., Eur J Biochem (1989) 182:639-647.
As the level of ACC synthase controls the production of ethylene, control of the level of this enzyme permits control of ethylene levels and thus regulation of the plant growth and development aspects that are controlled by ethylene. The availability of the ACC synthase gene, as provided by the invention herein, permits the construc tion of recombinant materials which permit such regula tion. Furthermore, the availability of ACC synthase makes possible large-scale production of an ethylene precursor useful in industrial production of this chemical and its products, such as ethanol.
Subsequent to the present invention, Van Der Straeten, D., et al. reported the cloning and sequences of cDNAs encoding ACC synthase from tomato (Proc Natl Acad Sci USA (1990) 87:4859-4863). Although the cDNA, which corresponded to an open reading frame of approximately 55 kd, produced a 55 kd peptide in E. coli, it is not clear from the data provided that this protein represents ACC
synthase.
Disclosure of the Invention The invention provides recombinant materials and techniques which permit control of the level of ACC
synthase in plants and portions thereof and also provides methods for large scale nonpetroleum-dependent production of ethylene. The recovery of a cDNA encoding the ACC
synthase of zucchini provides access to recombinant materials corresponding to alternate ACC synthases from zucchini as well as the range of ACC synthases in higher plants. This permits the control of plant development and activity in a wide variety of plant materials of commer cial interest.
Accordingly, in one aspect, the invention is directed to DNA in purified and isolated form consisting essen-tially of a DNA sequence encoding the enzyme ACC synthase of a higher plant. In other aspects, the invention is directed to expression systems effective in expressing the DNA encoding said ACC synthase, to recombinant hosts transformed with this expression system, and to methods of producing ACC synthase using these transformed hosts.

'" 2091 24 3 i In other aspects, the invention is directed to methods to control ACC synthase production using the coding sequences for ACC synthase in an antisense construct or by replacing the ACC synthase gene by a mutated form thereof.
In another aspect, the invention is directed to a novel method to isolate an inducible cDNA without necessity for the purified protein which it encodes.
In a broad embodiment the invention provides a DNA molecule comprising an expression system that is capable of generating, when contained in a plant host cell, a complementary RNA that is sufficiently complementary to an RNA transcribed from an endogenous ACC synthase gene to prevent synthesis of said endogenous ACC
synthase, wherein said expression system comprises the reverse transcript of said complementary RNA, said reverse transcript being operably linked to control sequences that effect it transcription into said complementary RNA.
In another embodiment the present invention provides a method for inhibiting ethylene production in a cell that normally produces ethylene, said method comprising culturing in vitro or in vivo a cell that has been modified to contain the DNA
molecule of any one of claims 1-3 under conditions wherein said complementary RNA is expressed.
In still a further embodiment the invention provides a transgenic plant that exhibits properties resulting from a lack of sufficient endogenous ACC synthase, wherein one of said properties is a delay of fruit ripening and wherein said transgenic plant has been modified to contain a DNA molecule comprising an expression system that generates an RNA complementary to RNA transcribed from an endogenous ACC synthase gene. The invention also provides the plasmid designated as pP032.
Brief Description of the Drawing's Figure lA shows a restriction map of two clones encoding the zucchini ACC synthase enzyme; Figure 1B shows the nucleotide and deduced amino acid sequence of one of these clones.

3a Figure 2A-2C show elution patterns in chromatographic steps in the purification of ACC synthase from zucchini.
Figure 3 shows the elution pattern of the final step in the purification of ACC synthase from zucchini.
Figure 4 shows an SDS-polyacrylamide gel of the fractions of Figure 3.
Figure 5 shows a restriction map of genomic clones obtained by hybridization to the cDNA encoding zucchini ACC synthase. Figure 5A shows the alignment of the retrieved clones with the position of the coding sequences on the genome; Figure 5B shows a restriction map of the sequences on the genome; Figure 5C shows the functional portions of the two zucchini ACC synthase genes CP-ACC lA
and CP-ACC 1B.
Figure 6 shows the complete nucleotide sequence and deduced amino acid sequence of the genomic clone repre-senting CP-ACC lA. Both control regions and coding regions are shown.
Figure 7 shows the complete nucleotide sequence and deduced amino acid sequence of the genomic clone repre-senting CP-ACC 1B. Both control regions and coding regions are shown.
Figure 8 shows the nucleotide and deduced amino acid sequence of a cDNA encoding the tomato ACC synthase.
Figure 9 shows a diagram and restriction map of WO 92/04456 ~ ~ ~ ~ ~ ~ PCT/US91 /0645 ~

several clones of the cDNA encoding tomato ACC synthase.
Figure 10 shows a comparison of the amino acid sequence of the ACC synthase encoded by the zucchini genomic sequence CP-ACC lA and the tomato genomic sequence ACC 2.
Figure 11 shows the pattern of genomic clones and functional diagrams thereof for the tomato genome containing coding and control sequences for LE-ACC lA, LE-ACC 1B, and LE-ACC 3.
Figure 12 shows the pattern of genomic clones and the organization of the gene for LE-ACC 2.
Figure 13 shows the complete genomic and deduced amino acid sequence of LE-ACC 2, including the control sequences.
Figure 14 shows a comparison of the deduced amino acid sequence from the two genomic zucchini clones and the four genomic tomato clones for ACC synthase.
Figure 15 shows the junction region and a restriction map of a bacterial expression vector for the production of tomato ACC synthase in bacteria.
Figure 16 shows the production of ACC by bacterial cultures transformed with the vector of Figure 15 in the presence and absence of the inducer IPTG.
Figure 17 is a half tone photograph of a two dimensional chromatographic gel of bacterial extracts wherein the bacterial culture is transformed with an expression vector for tomato ACC synthase having the coding sequence in the correct and incorrect orientations.
Figure 18 shows the construction of an expression vector for the tomato ACC-synthase gene oriented in the antisense direction.
Figures 19 and 20 show the ethylene production by tomato plants regenerated from tomato cotyledons trans formed with the vector of Figure 18 as a function of days from pollination.
Modes of Carryings Out the Invention The presence of ACC synthase as the controlling factor in ethylene production appears..to be universal in WO 92/04456 2 0 ~ i 2 4 ~ P~/US91/06453 higher plants. As a result of the recovery of cDNA
according to the invention, as described in Sato, T. and Theologis, A., Proc Natl Acad Sci USA (1989) 86:6621-6625, mailed tc subscribers September 11, 1989, and incorporated herein by reference, the presence of a gene family encod-ing a number of homologous, but different, ACC syntheses in zucchini has been shown. The various ACC syntheses control ethylene production at various locations in the differentiated plant, thus permitting separate control of, for example, fruit ripening and seed germination. In addition, the availability of this cDNA and the resulting genomic DNAs has permitted the recovery of DNAs encoding ACC syntheses in other plants, including, for example, tomato. While the various ACC syntheses are generally active in a variety of plant tissues, the DNAs are not completely homologous, and therefore the use of the genetic materials for control of synthesis, for example, using an antisense strategy, does not translate cross-species.
Definitions As used herein, "recombinant" refers to a nucleic acid sequence which has been obtained by manipulation of genetic material using restriction enzymes, ligases, and similar recombinant techniques as described by, for example, Maniatis et al. "Recombinant", as used in the present application, does not refer to naturally-occurring genetic recombinations.
As defined herein, "ACC synthase" includes all enzymes which are capable of catalyzing the conversion of AdoMet to ACC and methyl thioadenosine (MTA). The amino acid sequence of the synthase may or may not be identical with the amino acid sequence which occurs natively in higher plants. An example of such native sequence is shown in Figure 1 which occurs in the zucchini fruit (Cucurbita pepo). Naturally occurring allelic variants undoubtedly occur as well. Similar proteins are present in a wide variety of higher plants. In addition, artifi-cially induced mutations are also included so long as they -do not destroy activity. In general, conservative amino acid substitutions can be made for most of the amino acids in the primary structure as shown without affecting destruction of activity. Thus, the definition of ACC
synthase used herein includes these variants which are derived by direct or indirect manipulation of the dis-closed sequences.
It is also understood that the primary structure may be altered by post-translational processing or by subse l0 quent chemical manipulation to result in a derivatized protein which contains, for example, glycosylation substi-tuents, oxidized forms of, for example, cysteine or pro-line, conjugation to additional moieties, such as carriers, solid supports, and the like. These alterations do not remove the protein from the definition of ACC
synthase so long as its capacity to convert AdoMet to ACC
and MTA is maintained.
Thus, the identity of an enzyme as "ACC synthase" can be confirmed by its ability to effect the production of ethylene in an assay performed as follows: the enzyme to be tested is incubated with 200 ~,M AdoMet, 10 ~M pyridoxal phosphate, 40 ~g BSA in 20o mH Hepes buffer, pH 8.5 in a total volume of 600 ~1 at 30°C for 30 minutes, and the amount of ACC formed is assayed by conversion to ethylene using hypochlorite as described, for example, by Lisada, C.C., et al., Anal Biochem (1979) 100:140-145. While alternative forms of assessment of ACC synthase can be devised, and variations on the above protocol are certainly permissible, the foregoing provides a definite criterion for the presence of ACC synthase activity and classification of a test protein as ACC synthase.
The amino acid sequences for several ACC synthases in tomato and zucchini are shown in Figures 1, 8 and 14.
Preferred forms of the ACC synthases of the invention include those thus illustrated herein, and those derivable therefrom by systematic mutation of the genes. Such systematic mutation may be desirable to enhance the ACC
synthase properties of the enzyme, to enhance the WO 92/04456 _ 2 0 9 1 2 4 3 PCT/US91/06453 _ 7 _ characteristics of the enzyme which are ancillary to its activity, such as stability, or shelf life, or may be desirable to provide inactive forms useful in the control . of ACC activity in vivo.
As described above, "ACC synthase" refers to a - protein having the activity assessed by the assay set forth above; a "mutated ACC synthase" refers to a protein which does not necessarily have this activity, but which is derived by mutation of a DNA encoding an ACC synthase.
By "derived from mutation" is meant both direct physical derivation from a DNA encoding the starting material ACC
synthase using, for example, site specific mutagenesis or indirect derivation by syntheses of DNA having a sequence related to, but deliberately different from, that of the ACC synthase. As means for constructing oligonucleotide of the required length are available, such DNAs can be constructed wholly or partially from their individual constituent nucleotides.
As used herein, "higher plant" refers to those plants whose development and activity are controlled by ethylene.
These includes all common agricultural plants and various flowering species.
Initial Isolation of the ACC Synthase cDNA
The ACC synthase cDNA was isolated initially from Cucurbita pepo (zucchini) using a novel method which is applicable to inducible proteins. The method does not require pure protein in order to design probes or to prepare monoclonal antibodies; the method relies on the production of a cDNA expression library from induced tissue and identification of positive clones using an antibody preparation which has been purified by taking - advantage of the inducible nature of the protein. Thus, in general, the method comprises the steps of preparing - partially purified inducible protein of interest from the cells or tissue which have been induced for this production and preparing a composition of antibodies to these purified proteins. The composition of antibodies is prepared in a conventional manner by immunization of a -suitable mammal with a protocol designed to enhance the production of antiserum.
The first composition of antibodies, which will also contain antibodies to the protein contaminants in the preparation, is then purified to obtain a second antibody composition enriched in the antibodies immunoreactive for the desired protein. This enrichment is effected by reacting the first prepared composition with a protein extract from uninduced tissue. This will selectively remove those antibodies immunoreactive with background contaminants. This purified preparation of antibodies is then used to screen a cDNA expression library which has been prepared from tissue expressing the gene encoding the inducible protein. As the purified antibody preparation is immunospecific for this protein, identification of the positive clones is simplified. The application of this method to the recovery of cDNA encoding ACC synthase is described in detail in Example 1 herein. However, a similar method can be used to obtain the cDNA for any inducible protein, even without isolation and purification of the desired target protein.
Extension of the ACC Synthase Family The availability of the cDNA encoding zucchini ACC
synthase makes accessible both the multigene family which provides the variety of ACC synthases found in the same plant host-i.e., zucchini, as well as other cDNAs encoding ACC synthase from other species of higher plants and their corresponding multiple gene families. The cDNAs or portions thereof are used as probes to hybridize to the additional genomic or cDNA sequences by hybridization under standard conditions. Typical standard conditions of stringency include those set forth, for example, in Example 4. These recovered sequences can also be engineered to effect the expression of ACC synthase, to make modification which result in ACC synthase mutants, or "mutated ACC synthase" and to construct antisense vectors to control the production of indigenous ACC synthase.

WO 92/04456 2 0 ~ 12 4 3 pCT/US91/06453 ,..., Recombinant Production of ACC Synthase The availability of the ACC synthase gene permits its production in a variety of recombinant systems. Recombi-pant production of this enzyme in single cellular systems, including procaryotic and eucaryotic,systems, provides the tools for the recombinant production of ethylene nd ethanol, the products of the ACC synthesized. Large scale production of these chemicals can be effected by utilizing suitable large scale recombinant production of the ACC
synthase enzyme to effect the endogenous production of ACC
followed by chemical conversion of the ACC to ethylene and/or ethanol. In order to make such production economi-cally attractive, large scale production, such as in large algae cultures, is preferred. The ACC synthase can also be produced in transgenic plants both in enhanced amounts and in an antisense mode, as further set forth below, to control the aspects of plant development which are ethylene sensitive, and in particular, to delay plant genescence.
Accordingly, a variety of expression systems and hosts can be used for the production of this enzyme. A
variety of procaryotic hosts and appropriate vectors is known in the art; most commonly used are E. coli or other bacterial hosts such as ~ subtilis or Pseudomonas and typical bacterial promoters include the trp, lac, tac, and ~-lactamase promoters. A readily controllable, inducible promoter, the ~-phage promoter can also be used. A large number of control systems suitable for procaryote expres-sion is known in the art.
Similarly, a large number of recombinant systems have been developed for expression in eucaryotic hosts, including yeast, insect cells, mammalian cells, and plant cells. These systems are well characterized, and require the ligation of the coding sequence under the control of a suitable transcription initiating system (promoter) and, if desired, termination sequences and enhancers.
Especially useful in connection with the ACC synthase genes of the present invention are expression systems 209.243 _ -which are operable in plants. These include systems which are under control of a tissue-specific promoter, as well as those which involve promoters that are operable in all plant tissues.
5 Transcription initiation regions, for example, include the various opine initiation regions, such as octopine, mannopine, nopaline and the like. Plant viral promoters can also be used, such as the cauliflower mosaic virus 35S promoter. In addition, plant promoters such as 10 ribulose-1,3-diphosphate carboxylase, fruit-specific pro-moters, heat shock promoters, seed-specific promoters, etc. can also be used.
The cauliflower mosaic virus (CaMV) 35S promoter has been shown to be highly active in many plant organs and during many stages of development when integrated into the genome of transgenic plants including tobacco and petunia, and has been shown to confer expression in protoplasts of both dicots and monocots.
The CaMV 35S promoter has been demonstrated to be active in at least the following monocot and dicot plants with edible parts: blackberry, Rubus; blackberry/rasp berry hybrid, Rubus, and red raspberry; carrot, Daucus carota; maize; potato, Solanum tuberosum; rice, Oryza sativa; strawberry, Fraqaria x ananassa; and tomato, Lvcopersicon esculentum.
The nopaline synthase (Nos) promoter has been shown to be active in at least the following monocot and dicot plants with edible parts: apple, Malus pumila; cauli-flower, Brassica oleracea; celery, Apium graveolens;
cucumber, Cucumis sativus; eggplant, Solanum melong~ena;
lettuce, Lactuca sativa; potato, Solanum tuberosum; rye, Secale cereale; strawberry, Fraaaria x ananassa; tomato, Lycobersicon esculentum; and walnut, Jualans reg~ia.
Organ-specific promoters are also well known. For example, the E8 promoter is only transcriptionally activated during tomato fruit ripening, and can be used to target gene expression in ripening tomato fruit (Deikman and Fischer, EMBO J (1988) 7:3315; Giovannoni et al., The Plant Cell (1989) _1:53). The activity of the E8 promoter is not limited to tomato fruit, but is thought to be compatible with any system wherein ethylene activates biological processes.
Other organ-specific promoters appropriate for a desired target organ can be isolated using known proce-dures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, Phil, Trans R Soc London (1986) B314:343).
These mRNAs first isolated to obtain suitable probes for retrieval of the appropriate genomic sequence which retains the presence of the natively associated control sequences. An example of the use of techniques to obtain the cDNA associated with mRNA specific to avocado fruit is found in Christoffersen et al., Plant Molecular Bioloav (1984) 3:385. Briefly, mRNA was isolated from ripening avocado fruit and used to make a cDNA library. Clones in the library were identified that hybridized with labeled RNA isolated from ripening avocado fruit, but that did not hybridize with labeled RNAs isolated from unripe avocado fruit. Many of these clones represent mRNAs encoded by genes that are transcriptionally activated at the onset of avocado fruit ripening.
A somewhat more sophisticated procedure was described in Molecular Biology of the Cell, Second Edition (1989) pages 261-262, edited by Alberts et al., Garland Publishing Incorporated, New York. In this procedure, mRNAs enriched for organ-specific nucleic acid sequences were used to construct the cDNA library. This method was also applied to tomato by Lincoln et al. , Proc Natl Acad Sci (1987) 84:2793, and resulted in the production of an E8 cDNA clone.
The gene that encodes the organ-specific mRNA is then isolated by constructing a library of DNA genomic sequences from the plant. The genome library is screened with the organ-specific DNA clone, and the sequence is determined. The promoter is then isolated. These procedures are now considered to be routine and are described in detail in Sambrook et al., Molecular Cloninct:
A Labcratorv Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Either a constitutive promoter or a desired organ-specific promoter is then ligated to the gene encoding ACC
synthase or a mutated form thereof using standard tech-niques now common in the art. The expression system may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer ele-ments.
Thus, for expression in plants, the recombinant expression cassette will contain in addition to the ACC
synthase-encoding sequence, a plant promoter region, a transcription initiation site (if the coding sequence to be transcribed lacks one), and a transcription termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.
Sequences controlling eucaryotic gene expression have been extensively studied. Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs (bp) upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. By convention, the start site is called +1. Sequences extending in the 5' (upstream) direction are given negative numbers and sequences extending in the 3~ (down-stream) direction are given positive numbers.
In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element with a series of adenines surrounding the trinu-cleotide G (or T) NG (Messing, J. et al . , in Genetic Encti-neerinct in Plants, Kosage, Meredith and Hollaender, eds.
(1983) pp. 221-227). Other sequences conferring tissue specificity, response to environmental signals, or maximum efficiency of transcription may also be found in the promoter region. Such sequences are often found within 400 by of transcription initiation site, but may extend as far as 2000 by or more.
In the construction of heterologous promoter/struc tural gene combinations, the promoter is preferably posi tioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
As stated above, any of a number of promoters which direct transcription in plant cells is suitable. The promoter can be either constitutive or inducible.
Promoters of bacterial origin include the octopine syn-thase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids (Herrera-Estrella et al., Nature (1983) 303:209-213). Viral promoters include the 35S and 19S RNA promoters of cauli-flower mosaic virus (O'Dell et al., Nature (1985) 313:810-812). Plant promoters include the ribulose-1,3-diphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene and other genes in which expression in induced by ethylene may also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer, BO J (1988) 7:3315-3320 which is incorporated herein by reference.
In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
If the mRNA encoded by the structural gene is to be efficiently processed, DNA sequences which direct polyadenylation of the RNA are also commonly added to the vector construct (Albert and Kawasaki, Mol and Appl Genet, (1982) _1:419-434). Polyadenylation is of importance for 2~9~.243 -expression of the ACC synthase-encoding RNA in plant cells. Polyadenylation sequences include, but are not limited to the A~c~robacterium octopine synthase signal (Gielen et al., EMHO ~, (1984) 3:835:-846) or the nopaline synthase signal (Depicker et al., Mol and Appl Genet (1982) 1:561-573).
The resulting expression system or cassette is ligated into or otherwise constructed to be included in a recombinant vector which is appropriate for higher plant transformation. The vector will also typically contain a selectable marker gene by which transformed plant cells can be identified in culture. Usually, the marker gene will encode antibiotic resistance. These markers include resistance to 6418, hygromycin, bleomycin, kanamycin, and gentamicin. After transforming the plant cells, those cells having the vector will be identified by their ability to grow on a medium containing the particular antibiotic. Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range procaryotic origin of replication is included.
A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct. Suitable procaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.
Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
For instance, in the case of Ag~robacterium transformation, T-DNA sequences will also be included for subsequent transfer to plant chromosomes.
In addition, vectors can also be constructed that contain in-frame ligations between the sequence encoding the ACC synthase protein and sequences encoding other molecules of interest resulting in fusion proteins, by techniques well known in the art.
When an appropriate vector is obtained, transgenic plants are prepared which contain the desired expression WO 92/04456 2 ~ g 12 ~3 PCT/US91/06453 system. A number of techniques are available for transformation of plants or plant cells. All types of plants are appropriate subjects for "direct"
transformation; in general, only dicots can be transformed using Agrobacterium-mediated infection.
In one form of direct transformation, the vector is microinjected directly into plant cells by use of micro-pippettes to mechanically transfer the recombinant DNA
(Crossway, Mol Gen Genetics (1985) 202:179-185). In another form, the genetic material is transferred into the plant cell using polyethylene glycol (Krens, et al., Nature (1982) 296:72-74), or high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, is used (Klein, et al., Nature (187) 327:70-73). In still another method protoplasts are fused with other entities which contain the DNA whose introduction is desired. These entities are minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley, et al., Proc Natl Acad Sci USA (1982) 79:1859-1863.
DNA may also be introduced into the plant cells by electroporation (Fromm et al., Proc Natl Acad Sci USA
(1985) 82:5824). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide and regenerate.
For transformation mediated by bacterial infection, a plant cell is infected with Actrobacterium tumefaciens or A. rhizogenes previously transformed with the DNA to be introduced. Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefciens) and hairy root disease (A. rhizogenes). The plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by X091243 _ the bacteria. The bacterial genes responsible for expres sion of opines are a convenient source of control elements for chimeric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue.
Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A.
tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome (Schell, J., Science (1987) 237:1176-1183). Ti and Ri plasmids contain two regions essential for the production of transformed cells. One of these, named transferred DNA (T-DNA), is transferred to plant nuclei and induces tumor or root formation. The other, termed the virulence (vir) region, is essential for the transfer of the T-DNA but is not itself transferred. The T-DNA
will be transferred into a plant cell even if the vir region is on a different plasmid (Hoekema, et al., Nature (1983) 303:179-189). The transferred DNA region can be increased in size by the insertion of heterologous DNA
without its ability to be transferred being affected. Thus a modified Ti or Ri plasmid, in which the disease-causing genes have been deleted, can be used as a vector for the transfer of the gene constructs of this invention into an appropriate plant cell.
Construction of recombinant Ti and Ri plasmids in general follows method typically used with the more common bacterial vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to "shuttle vectors," (Ruvkum and Ausubel, Nature (1981) 298:85-88), promoters (Lawton et al., Plant Mol Biol (1987) 9:315-324) and structural genes for antibiotic resistance as a selection factor (Fraley et al., Proc Natl Acad Sci (1983) 80:4803-4807).
There are two classes of recombinant Ti and Ri plasmid vector system now in use. In one class, called "cointegrate," the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and tans-acting elements required for plant transformation as, for example, in the pMLJi shuttle vector of DeBlock et al., BO J (1984) 3:1681-1689 and the non-oncogenic Ti plasmid pGV2850 described by Zambryski et al., EMBO J
(1983) 2:2143-2150. In the second class or "binary"
system, the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBINl9 shuttle vector described by Bevan, Nucleic Acids Research (1984) 12:8711-8721 and the non-oncogenic Ti plasmid PAL4404 described by Hoekma, et al., ature (1983) 303:179-180. Some of these vectors are commercially available.
There are two common ways to transform plant cells with Aqrobacterium: co-cultivation of Ag~robacterium with cultured isolated protoplasts, or transformation of intact cells or tissues with Ag~robacterium. The first requires an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cul tured protoplasts. The second method requires (a) that the intact plant tissues, such as cotyledons, can be transformed by Aqrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
Most dicot species can be transformed by Agrobacterium as well as species which are a natural plant host for Agrobacterium are transformable in vitro.
Monocotyledonous plants, and in particular, cereals, are not natural costs to Ag~robacterium. Attempts to transform them using Agrobacterium have been unsuccessful until recently (Hooykas-Van Slogteren et al., Nature (1984) 311:763-764). However, there is growing evidence now that certain monocots can be transformed by Actrobacterium.

WO 92/04456 ~ ~ ~ ~ PCT/US91 /06453 Using novel experimental approaches cereal species such as rye (de la Pena et al., Nature (1987) 325:274-276), maize (Rhodes et al., Science (1988) 240:204-207), and rice (Shimamoto et al., Nature (1989) 338:274-276) may now be transformed.
Identification of transformed cells or plants is generally accomplished by including a selectable marker in the transforming vector, or by obtaining evidence of successful bacterial infection.
Plant cells which have been transformed can also be regenerated using known techniques.
Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co. New York, 1983); and Vasil I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando,, Vol. I, 1984, and Vol. II, 1986). It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugar-cane, sugar beet, cotton, fruit trees, and legumes.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted.
Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regene-ration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
A large number of plants have been shown capable of regeneration from transformed individual cells to obtain transgenic whole plants. For example, regeneration has been shown for dicots as follows: apple, Malus pumila;
blackberry, Rubus, Blackberry/raspberry hybrid, Rubus, red raspberry, Rubus; carrot, Daucus carota; cauliflower, Brassica oleracea; celery, Anium graveolens; cucumber, Cucumis sativus; eggplant, solanum melongena; lettuce, Lactuca sativa; potato, Solanum tuberosum; rape, Brassica us; soybean (wild), Glycine canescens; strawberry, Frag~aria x ananassa; tomato, Lvcopersicon esculentum;
walnut, Jug~lans reg~ia; melon, Cucumis melo; grape, Vitis vinifera; mango, Mangifera indica;
and for the following monocots: rice, Oryza sativa;
rye, Secale cereale; and maize.
In addition, regeneration of whole plants from cells (not necessarily transformed) has been observed in:
apricot, Prunus armeniaca; asparagus, Asparagus offici nalis; banana, hybrid Musa; bean, Phaseolus vulgaris;
cherry, hybrid Prunus; grape, Vitis vinifera; mango, Manqifera indica; melon, Cucumis melo; ochra, Abelmoschus esculentus; onion, hybrid Allium; orange, Citrus sinensis;
papaya, Carrica papaya; peach, Prunus ~ersica and plum, Prunus domestica; pear, Pvrus communis; pineapple, Ananas comosus; watermelon, Citrullus vulg~aris; and wheat, Triticum aestivum.
The regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner.
After the expression cassette is stably incorporated into regenerated transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
The plants are grown and harvested using conventional procedures.
Antisense Expression When the ACC synthase coding sequence is placed in correct orientation in the expression systems described above, the ACC synthase protein is produced. However, when placed in the opposite orientation, the expression vector WO 92/04456 ~ ~ ~ ~~ ~ PCT/US91/06453 has an antisense effect which can interfere with the indigenous production of this enzyme. Antisense tech-nology can work at a variety of levels including hybridi-zation to a messenger RNA encoding the ACC synthase, hybridization to single-stranded intermediates in the production of this mRNA, or triplex formation with the DNA
duplex which contains the ACC synthase genes. All of these modalities can be employed in effecting antisense control of ACC synthase production.
As shown in Example 8 below, ripening of tomato fruit can be controlled and inhibited by suitable antisense expression of the ACC synthase coding sequence supplied in a vector under the control of the cauliflower 35S
promoter. Other properties which are controlled by ethylene can also be influenced by appropriate choice of control systems and/or the particular AC synthase encoded.
It is further shown below that the active form of ACC
synthase in higher plants is a dimer. By supplying a mutated form of ACC synthase monomer, a decoy can be pro duced to obtain inactive monomer and thereby regulate the levels of ACC synthase in the plant. An additional embodiment of the invention involves the mutated ACC
synthase and expression systems therefor.
The following examples are intended to illustrate but not to limit the invention.
Example 1 Recovery of Zucchini ACC Synthase cDNA
A cDNA encoding ACC synthase in zucchini fruit was recovered as follows:
Slices 1 mm thick were prepared from zucchini fruits of the species Cucurbita pepo. To induce production of ACC synthase, slices were incubated for 18-24 hours in induction medium (50 ~,M potassium phosphate buffer, pH
6.8; 0.5 mM indole acetic acid (IAA); 0.1 mM benzyl adenine (BA); 50 mM LiCl; 0.6 mM aminooxyacetic acid (AOA); and 50 ug/ml chloramphenicol. (Uninduced tissue was prepared in a similar manner in 50 mM phosphate buffer, pH 6.8.) WO~/04456 PCT/US91 /06453 - 21 _ 209 1 24 3 Poly(A') RNA (mRNA) was isolated from 18-hr tissue prepared as described above, and in vitro translated in a wheat germ lysate as described by Theologis, A., et al., J_ Mol Biol (1985) 183:53-68, in the presence of labeled methionine (greater than 1,000 Ci/~mol) to verify the presence of ACC synthase encoding mRNA. A cDNA library was prepared in ~gtli as described by Huynh, T.V., et al., in "DNA Cloning Techniques," Glover, E., ed. (1985) IRL
Press, Oxford, x:49-88. The insert sizes were 200-500 bp.
The library was screened with purified ACC synthase anti-serum prepared as follows:
The antisera were prepared to 1500-fold purified ACC
synthase preparations. Purified ACC synthase can be prepared from tissue homogenates sequentially bound to and eluted from Butyl Toyopearl* (Toyo Soda Tokyo), SP-Sephadex*, and QAE-Sephadex*. (Higher purification can be obtained by subsequent chromatography sequentially through columns containing Butyl Toyopearl, Sephacryl S-00*, Bio Gel-Ht*, and finally FPLC mono-Q*. The application of all of the following steps results in approximately a 6000-fold purification.) The antibodies are prepared in New Zealand white rabbits by immunization protocols involving four immunizations at three-week intervals with 5000 nmol of ACC
synthase activity/hr (specific activity 1500 nmol of, ACC/hr/mg protein obtained from the Bio Get-HT column).
Crude antiserum (2 ml) was purified by incubation with 10 ml Sepharose 4B* coupled with soluble proteins from intact noninduced Cucurbita fruit. This step removed antibodies immunoreactive with protein other than ACC synthase.) Sixty-six immunoreactive clones were isolated by screening 1.4 x 105 ~gtii recombinant clones with the purified antiserum. Upon rescreening, only 30 were, in fact, positive. Southern analysis showed that 19 clones represented the ACC synthase mRNA. One selected clone, pACCl, has an open reading frame encoding. a 55.8 kd polypeptide. Another intensely immunoreactive clone, pACC7, was much shorter. Figure lA shows a restriction map of pACCl and pACC7; Figure 1B shows the complete *Trade-mark VND 92/044 g ~ ~ ~ 3 PCT/ US91 /06453 nucleotide sequence and deduced amino acid sequence for these clones.
As shown in Figure 1B, pACC7 is identical to a portion of the sequence of pACCl. The open reading frame encodes a protein of 493 amino acids, corresponding to a 55.779 kd polypeptide.
The positive clones from the ~gtll library could also be used to prepare further purified antiserum for immuno-blotting as follows:
The positive cones from the expression library was plated on E. coli strain Y1090 to obtain 105 plaque-form-ing units per 90-mm plate. Dry nitrocellulose filters presoaked in 10 mM isopropyl ~i-D-thiogalacto-pyranoside (IPTG) were laid on the lawn after incubation for two hours at 42°C and then incubated for an additional four hours at 37°C.
The filters were then soaked for 30 minutes in TBST
(50 mM Tris HC1, pH 8.0; 0. 14M NaCl; 0.05% Tween 20) ; 2%
milk protein and then tested for ACC synthase expression by treating with 5 ml of diluted (1:500) purified ACC
synthase antiserum (see below) per filter for two hours.
After washing five times at 10 minutes each with TBST, bound antibody was eluted by shaking for three minutes at room temperature with 0.2M glycine hydro chloride buffer, pH 2.3, containing 1% milk protein. The antibody solution was neutralized and used for immuno-blotting.
Example 2 Purification of Native ACC Synthase From Cucurbita ACC synthase was purified 6000-fold from induced Cucurbita homogenates according to a multistep protocol as shown below. Various buffers used in the purification are as follows:
Buffer A: Tris-HC1 100 mM, pH 8.0, EDTA 20 mM, pyridoxal phosphate 10 ~,M, PMSF 0.5 mM, ~i-mercaptoethanol 20 mM; Buffer B: Tris-HC1 20 mM, pH 8.0, EDTA 10 mM, pyridoxal phosphate 10 ~,M, DTT 0.5 mM; Buffer C: Na-acetate 20 mM, pH 6.0, pyridoxal phosphate 10 ~M, EDTA 10 WO 92/04456 2 p 912 4 3 PCT/US91/06453 mM, DTT 0.5 mM; Buffer D: K-phosphate 10 mM, pH 8.0, pyridoxal phosphate 10 ~,M, EDTA lmM, DTT 0.5 mM; Buffer E:
Tris-HC1 20mM, pH 8.0, pyridoxal phosphate 5 ACM, EDTA 1 mM, DTT 0.5 mM; Buffer F: Hepes-KOH 500 mM, pH 8.5, pyridoxal phosphate 40 ~,M, BSA 400 ~g/ml.
All operations were performed at 4°C. Chroma-tographic elutions were assayed for ACC synthase activity and by absorption at 280 nm.
Ten kg of Cucurbita slices incubated for 24 hr in induction medium were chilled with liquid Nz and homoge nized in batches of 2 kg with 2 liters of buffer A plus 200g of pol~y~inylpolypyrrolidone in a one gallon Waring~
blender for 1 min at medium speed. The homogenate was centrifuged at 17,000 x g for 30 min. The supernatant was filtered through one layer of microcloth and one layer of nylon cloth (30 ~,m mesh).
Butyl Tovopearl Fractionation Solid ammonium sulfate was added slowly to the stirred supernatant above to achieve 40% saturation. The supernatant solution was stirred for 15 min and 300 ml of packed Butyl Toyopearl 650 M hydrophobic affinity matrix, previously equilibrated with buffer B saturated to 40%
with ammonium sulfate, were added. The suspension was occasionally stirred for an additional 30 min. The matrix was recovered by filtration through one layer of nitex nylon cloth (30 ~m mesh) and the solution was squeezed out by hand. Subsequently, the matrix was placed in a vacuum filter with two sheets of Whatman filter paper #1 and washed with 500 mil of buffer B containing 40% ammonium sulfate. The adsorbed proteins were eluted from the matrix by washing (twice) with 750 ml of buffer B, batch-wise. The combined eluates were dialyzed three times against 10 liters of buffer B for 36 hr.
SP-Sephadex Fractionation The dialyzed fraction above was clarified by centri-fugation at 17,000 x g for 30 min. The volume was adjusted to 4 liters with buffer B and the pH was brought to pH 6.0 with 5% acetic acid. Two liters of packed SP-Sephadex C-50 equilibrated with buffer C were added and the suspension was stirred for 60 min. The matrix was recovered by filtration through two sheets of Whatman filter paper #1 and washed with 2 liters of buffer C. The adsorbed proteins were eluted twice with 1 liter of buffer B containing 1M KC1, batchwise. The eluant was recovered by suction through #1 Whatman filter paper and solid ammonium sulfate was added to achieve 40% saturation.
Subsequently 100 ml of Butyl Toyopearl-packed matrix equilibrated with buffer B/40% ammonium sulfate was added to the eluate. The suspension was stirred for 30 min and the matrix was collected by filtration through a layer of Nitex nylon cloth (30 ~Cm mesh). The matrix was resus-pended in a small volume of buffer B/40% ammonium sulfate and poured in a column (2.5 x 20 cm). The adsorbed pro-teins were eluted with buffer B, and the flow rate of the column was under gravity. Fractions with high AZgo were pooled and dialyzed overnight against 4 liters buffer B
with three buffer changes during the course of dialysis.
QAE-Se~hadex Fractionation Four hundred ml of packed QAE-Sephadex equilibrated with buffer B were added to the dialyzate from the SP-Sephadex fractionation and the suspension was stirred gently for 60 min. The matrix was recovered by filtration through a layer of miracloth in a filtration apparatus and washed with 500 ml of buffer B to remove unadsorbed proteins. The matrix was resuspended in a small volume of buffer B and poured into a column (4 x 30 cm). The proteins were eluted with buffer B containing 0.2M KC1.
Butyl Toyopearl Chromatography Solid ammonium sulfate was added to the eluate (-100 ml) to achieve 40% saturation and the solution was kept at 4°C for at least 4 hr. The suspension was centrifuged at 30,000 x g for 30 min and the supernatant was applied on a Butyl Toyoperl column (1.5 x 14 cm) equilibrated with buffer B/40% ammonium sulfate. After all the protein solution was passed through the column, it was eluted with a 400 ml linear gradient: 40 to o% ammonium sulfate in WO 92/04456 2 0 912 4 3 P~/US91/06453 buffer B with a flow rate of 1 ml/min.
Figure 2A shows the elution pattern. Solid ammonium sulfate caas added to enzymatically active fractions to achieve 80$ saturation and the solution was incubated at 4°C for at least 4 hr. The precipitate was collected by centrifugation at 30, 000 x g for 30 min at 4 °C and dis-solved in 3 ml of buffer D.
Sephacryl S-300 Chromatography The resulting protein solution from above was applied to a column (2.5 x 100 cm) of Sephacryl S-300 equilibrated with buffer D. The column was eluted with buffer D at a flow rate of 0.5 ml/min. Figure 2B shows the elution pattern.
Bio Gel-HT Chromatogram Active fractions from the Sephacryl step were com-bined and applied on a Bio Gel-HA column (0.75 x 14 cm) equilibrated with buffer D. The column was washed with buffer D until AZao = 0 and it was then eluted with a 200 ml linear gradient: 10-1C0 mM potassium phosphate in buffer D with a flow rate of 0.1 ml/min. Figt:re 2C shows the elution pattern. The active fractions were collected and concentrated with a Centricon 30 filtration apparatus, concomitantly the bufF~~r of the concentrated protein solution was changed to buffer E.
FPLC Mono-O Chromatograrh~
The concentrated active fractions (-0.5 ml) from the.
Bio Gel-HT column were applied to a mono-Q H5/5 column.
The column was washed with buffer E containing O.1M KC1 until. A~FO = 0. The column then was eluted with a 15 ml linear gradient: 0.1 to 0.4M KC1 in buffer E. The flow rate of the gradient was 0.5 ml/min. Finure 3 shows the elution pattern.
Table 1 shows the overall purification sequence and the increase in specific activity with each successive step. The overall process results in a 6000-fold purifi cation with a recovery of 7.5%. The enzyme has a specific activity of 35,590 nmol ACC produced /hr/mg of protein.

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SDS-PAGE conducted on fractions 16 and 17 from the mono Q column, which have the highest ACC synthase activity, indicated that the protein was not completely pure. (See Figure 4) However, it was demonstrated that the ACC synthase activity resided in the 46 kd band. The electrophoresis was conducted by applying 7.5 ml of the eluted fractions mixed with an equal volume of 2 x SDS
loading buffer to a 10% polyacrylamide gel and silver staining. To determine the band containing ACC synthase activity, similar gels were run wherein the gels were cut into 3 mm thick slices and the ACC synthase activity was determined in half the slices; the other half were stained with silver.
The purified ACC synthase was also subjected to size exclusion chromatography o Sephacryl S-300. In this protocol, the ACC synthase eluted as an 86 kd species.
This suggests that the Cucurbita ACC synthase consists of two identical 46 kd subunits. Further characterization showed that the pH optimum for ACC synthase activity is 9.5, and the isoelectric point is estimated at 5 using mono-P H 5/20 FPLC column chromatography. The Km for AdoMet is 16.7 mM, and pyridoxal phosphate is a cofactor.
The enzyme is stable at -20°C or -80°C for over a year.
Example 3 Isolation of Zucchini Genomic Clones Encodinq ACC
Synthase Four-day-old etiolated frozen zucchini seedlings were homogenized in 15% sucrose, 50 mM Tris-HC1, pH 8.5, 50mM
EDTA-Na3, 0.25M NaCl. The nuclei were pelleted by centri-fugation at 4,000 rpm for 10 min at 4°C and nuclear DNA
was isolated by CsCl ethidium bromide equilibrium density gradient centrifugation. The recovered DNA was partially digested with Sau3A and electrophoretically separated on 0.5% low melting agarose.
DNA corresponding to 20 kb in size was ligated into EcoRI/BamHI cut EMBL 3a (Frischauf, A.M., et al., J Mol Biol (1983) 170:827-842; Raleigh, E.A., et al., Proc Natl Acad Sci USA (1986) 83:9070-9074; Maniatis, T., et al., Molecular Cloninct (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The ligation mixture was pack-aged and the library was screened without amplification by plating on E. coli strain K802 and screening with nick translated ACC1 cDNA (the full length zucchini cDNA clone) as described by Benton, D., et al., Science (1977) 196:179-183. The isolated genomic sequences were mapped with restriction endonucleases and the appropriate DNA
fragments which hybridize to the ACC1 cDNA were subcloned into the pUCl8 and pUCl9 plasmids.
The results after restriction analysis of the various genomic clones recovered is shown in Figure 5A-C. As shown in the figure, two genomic clones reside on the same DNA strand, but are oriented in opposite directions.
CP-ACC lA and CP-ACC 1B each contain four introns. The complete genomic sequences of these clones are shown in Figures 6 and 7 respectively. As shown in Figures 6 and 7, the entire upstream regulatory sequences are encoded in the clones.
Example 4 Retrieval of Tomato cDNA Encoding ACC Synthase Lycopersicon esculentum c.v. Rutgers was grown from seeds throughout the year in a greenhouse using protocols to ensure freedom from tobacco mosaic virus. The fruit was frozen and total RNA was isolated using the procedure of Chomcynzki, P., et al., Anal Biochem (1987) 162:156-159. Approximately 5 gm of powdered frozen fruit tissue were used. Poly (A)+ RNA was isolated using oligo (dT) cellulose chromatography as described by Theologis, A., et al. J Mol Biol (1985) 183:53-58, and a cDNA library was constructed into ~gtl0 as described by Huynh, T.V., et al.
cDNA Cloning TechnicLues: A Practical Approach (1985) (Glover, D.M. ed.), IRL Press, London, 49-78. cDNAs greater than 500 by were inserted into the EcoRI site of the C1 repressor gene. The packaged DNA was plated on C600 HFL, a derivative of C600, to select for phage-containing inserts.
Approximately 106 plaque forming units of the ~gtl0 recombinant phage were plated to a density of 1 x 104 per 85 mm petri dish using C600. After transferring to nitrocellulose filters as described above, prehybridization-and hybridization were performed at 37°C
with gentle agitation in 30% formamide, 5X SSPE (iX SSPE
is 0.1 8M NaCl, 10 mM sodium phosphage, pH 7.0, 1 mM
sodium EDTA), 5X BFP (iX BFP is 0.02% w/v bovine serum albumin, 0.02% polyvinyl pyrrolidone (Mr=360 kd), 0.02%
Ficoll (M,.=400 kd) , 100 mg/ml heat denatured salmon sperm DNA, and 0.1% SDS).
The gel purified 1.8 kb EcoRI fragment of the zucchini pACCl prepared in Example 1 was labeled to a specific activity of 2 x 10$ cpm/mg using random hexamer printing and a-32P dCTP as described by Feinberg, A.P., et al., Anal Biochem (1983) 132:6-13. The labeled probe was separated from starting material and used to probe the ~gtl0 library.
The probe was denatured with 0.25 volumes 1M NaOH for 10 minutes at room temperature and neutralized with 0.25 volume 2M Tris HC1, pH 7.2 and then added to the hybridi zation mixture at 1 x 106 cpm/ml.
After 24 hr hybridization, the filters were washed once in 30% formamide, 5X SSPE, 0.1% SDS at 37°C for 20 min and then four times in 2X SSPE, 0.1% SDS at 37°C for 20 min. The final wash was in 2X SSPE at 50°C for 10 min.
After washing, the filters were air dried, covered with Saran wrap and exposed at -70°C to X-ray film.
Using this hybridization, a full length cDNA from tomato, designated ptACCl, was recovered. The complete cDNA sequence of the ACCT of tomato, designated ptACCl, is shown in Figure 8. Additional clones were recovered using 2 x 106 cpm of the labeled 0.55 kb HindIII/EcoRI fragment at the 3' end of ptACCi.
Hybridization conditions were employed using 2 x 105 pfu of the ~gtl0 library on C600 wherein nitrocellulose platelets were prehybridized at 42°C and 50% formamide, 5X
SSPE, 5X BPF, 500 mg/ml heat denatured salmon sperm DNA
for 12 h. The filters were then hybridized for 18 hr at --M.
~zo~nz43 42°C with probe in 50% formamide, 5 X SSPE, 1X BFP, 100 mg/ml heat denatured salmon sperm DNA with this probe.
The filters were washed at 42°C twice for 30 min in 50%
formamide, 5X SSPE, 0.2% SDS, and then twice for 30 min i~
O.1X SSPE at 42°C. A number of additional clones were retrieved using the above referenced portion of ptACCl as shown in Figure 9.
A comparison of the amino acid sequences of the zucchini and tomato cDNA encoded ACC synthases is shown in Figure 10. As shown, considerable homology exists between these sequences, but they are not identical.
Example 5 Recovery of Tomato Genomic DNA Encoding ACC Synthase Genomic DNA was isolated from etiolated Rutgers seed lings using a modification of the method described by Davis, R.W., et al. Meth Enzymol (1980) 65:404-411.
Briefly, seedlings were grown on moist filter paper in the dark for 5 days at 22°C. Fifty g frozen hypocotyl and cotelydon tissue, seed coat removed, was ground in a coffee grinder. The powdered tissue was added to 200 ml of ice cold extraction buffer (50 mM Tris-HC1, pH 8.0, 50 mM NaEDTA, 0.25 M NaCl, 15% sucrose (w/v)) and homogenized on ice using a hand held glass-glass homogenizer. The nuclei were pelleted at 2000 x g for 10 min at 4°C. The crude nuclei we resuspended in 100 ml of cold extraction buffer without the salt. To lyse the nuclei, 10 ml of 10%
Nasarcosine was added, the suspension was gently inverted and incubated on ice for 10 min, then 120 g of CsCl was added and dissolved by gently shaking. To remove debris the solution was centrifuged at 26,OOOxg for 20 min at 4°C
and the supernatant was decanted through Miracloth.
Ethidium bromide (10 mg/ml) was added to a final concentration of 0.4 mg/ml and the density of the solution was adjusted to 1.55 g/ml. Equilibrium centrifugation was carried out in a Beckman Ri70 rotor at 40,000 rpm for 48 hr at 20°C. The DNA was further purified by a second round of equilibrium centrifugation in a VTi65 rotor at 50,000 rpm for 16 hr at 20°C. Ethidium bromide was W0,~.2/04456 ' PCT/US91/06453 extracted from the DNA with isopropanol saturated with water containing 5 M NaCl and the DNA was dialyzed twice against 5000 volumes of TE (pH 7.5) to remove the CsCl.
The tj~ical yield was 15 ~Cg/g fresh weight tissue.
Two genomic libraries were constructed, one with 15-23 kb Sau3A partially digested DNA in ~EMBL3 and another with 6-8 kb DNa after complete HindIII digestion into X2001. For the library constructed in ~EMBL3, 100 ~cg of genomic DNA was digested with 1.5 units of Sau3A at 37°C
in 300 ~cl of medium salt buffer (MSB) plus 2 mM
dithiothreitol (DTT) (1X MSB is 10 mM Tris-HC1, pH 7.5, 50 mM NaCl, l0 mM MgSO,). One third of the reaction was removed at 7.5 min, at 10 min and at 12.5 min. At each time point digestion was stopped by adding O.i volume 0.5 NaEDTA, pH 8.0 and storing on ice. The DNA was size fractionated in a 0.5% low melting temperature agarose gel by electrophoresis at 0.8 Volts/cm for 24 h. The agarose gel electrophoresis buffer was 1X TAE, 40 mM Tris-HOAc, pH

8.0, 2 mM NaWDTA. The gel was soaked at room temperature for 3 hr in 1X TAE buffer containing 0.3 M NaCl. DNA was visualized with 365 nm ultraviolet light and the 15-23 kb side range was excised. The agarose was melted at 65°C
for 15 min and extracted twice the TE (pH 7.5)-saturated phenol, prewarmed to 37°C. The aqueous phase was extracted three times with ether and two volumes of EtOH
were added. After overnight at -20°C the DNA was collected by centrifugation and dissolved in TE, pH 7.5 Two ~cg of EMBL3 arms and 2~g size selected DNA were com-bined in a final volume of 6 ~cl, 1 ul of 10X ligase buffer (1X ligase buffer is 66 mM Tris-HCl, pH 7.5 5 mM MgCl2) was added and the cohesive ends annealed at 42°C for 15 min. The mixture was quickly cooled on ice and l.~c~. each of 10 mM ATP and 50 mM DTT was added. The reaction was initiated with l~Cg (8 units) of T4 DNA ligase and incubated overnight at 14°. One third of the ligation mix was pacYaged with Gigapak Gold* (Strategene) according to the manufacturer. Approximately 1 x 106 pfu were obtained when titered on C600.
;~ *Trade-mark WO 92/04456 ~ ~ ~ ~ PCT/US91 /06453 For the HindIII library, 200 ~g of genomic DNA was digested in 3.6 ml 1X MSB with 400 units of enzyme for 4 hr at 37°C. The DNA was separated on a 0.08% low melting temperature agarose gel and DNA in the 6-8 kb size range was isolated as described above. One ~g of this DNA was ligated to 0.5 ~g of X2001 arms as described above in a final volume of 10 ~1. One third of this ligation was packaged with Gigapak Gold and 5 x 10' pfu were obtained when plated on K802.
A BglII complete digest library and an MboI partial digest library of genomic DNA from tomato cultivar VF36, both in EMBL4, were provided by C. Corr and B. Baker.
These libraries and the HindIII complete digest library in X2001 were plated on the host K802 and probed at high stringency with the ptACCl cDNA as described above to obtain clones corresponding to the cDNA. Clones corre-sponding to other genes were obtained by probing the Sau3A
partial digest library in EMBL3 with the cDNA at low stringency. In two separate screenings, phage were plated on the hosts C600 or TC410, lifted and fixed to nitro-cellulose filters as described above. Low stringency prehybridization and hybridization were done in 30%
formamide, 5X SSPE, 5X BFP, 100 ~g/ml denatured salmon sperm DNA, 0.2% SDS at 37°C for 18 h each. Probe was used at a concentration of 106 cpm/ml. Washing was done twice for 20 min in 30% formamide, 5X SSPE, 0.2% SDS at 37°C, and twice for 20 min in 2X SSPE, 0.2%, SDS at 42°C. The filters were exposed to X-ray film as above for 48 h.
For restriction enzyme digestions of ~ clones, 2.5 ~,g of phage DNA was digested in 50 ~,1 of high salt buffer (HSB) (1X HSB is 100 mM NaCl, 50 mM Tris-HC1 (pH 7.5), 10 mM MgS04) with the appropriate enzymes) . For genomic DNA
gel blots, 7.15 ~Cg of genomic DNA was digested in 100 ~1 of 1X HSB with 80 units of EcoRI and HindIII or 40 units of BglII, at 37°C for 6 h. Digested DNAs were loaded on 1 cm thick, 0.8% agarose gels and electrophoresed at 3 V/cm in iX TAE buffer containing 0.5 ~,g/ml ethidium bromide.
After electrophoresis the gel was photographed, the DNA

WO~,/04456 PCT/US91 /06453 was nicked with two 15 min treatments of 0.25 M HC1, denatured with two 20 min treatments of 0.5 M Tris-HC1 (pH
7.5), 1.5 M NaCl and neutralized with two 20 min treat-ments cf 0.5 M Tris-HC1 (pH 7.5), 1.5 M NaCl. The nucleic acids were transferred in 20X SSPE to a Nytran nylon membrane.
After transfer was complete the nucleic acids were fixed with 1.2 joules of 254 nm ultraviolet radiation.
The membranes were prehybridized in 50 ml of 50%
formamide, 5X SSPE, SX BFP, 1.0% SDS and 100 ~Cg/ml heat denatured salmon sperm DNA at 42°C for 12 h.
Hybridizations were carried out in 30 ml of 50% formamide, 5X SSPE, 1X BFP, 10% dextran sulfate (Mr=400 kd),-- 0.2%
SDS, and 50 ~Cg/ml heat-denatured salmon sperm DNA at 42°C
for 18 h. Filters with genomic DNA were hybridized with 2.0 x 106 cpm/ml, whereas filters with ~ DNA were hybridized with 5 x 105 cpm/ml of random hexamer labeled 1.8 kb ptACCl cDNA. After hybridization the membranes were washed two times for 20 min at 55°C in O.1X SSPE and 0.2% SDS, dried, wrapped in plastic wrap and placed under Kodak XR-5 X-ray film. 1~ DNA gel blots were exposed for 12-24 hr at -70° with an intensifier screen.
The clones corresponding to four different genomic clones recovered from tomato are shown in Figures 11 and 12. Figure 11A shows a series of three genomic clones which were identified to three separate. genes LE-ACC lA;
LE-ACC 1B; and LE-ACC 3; Figure 12 shows genomic clones which were identified with LE-ACC 2. Figure 13 shows the complete nucleotide sequence of LE-ACC 2.
Figure 14 compares the amino acid sequences encoded by the six genomic clones recovered--two from zucchini and four from tomato. Again, conserved sequences are found and there is considerable homology; however, there are numerous differences in sequence.
Example 6 Expression of Zucchini and Tomato cDNA in E. coli The pACCl from zucchini was subcloned into the EcoRI
site of the expression vector pKK223-3 (DeBoer, H.A., et *Trade-mark 2pg~.243 _ al, Proc Natl Acad Sci USA (1983) 80:21-25) and introduced into E. coli stain JM107. Transformants were grown in LB
medium in the presence of ampicillin (50 mg/ml) at 37°C
for 4 h. IPTG was added to 1 mM and the cultures were incubated for 2 hr at 37°C. ACC synthase activity and ACC
formation were assayed. When the 1.7 kb EcoRI cDNa fragment was inserted into pKK2233-3 in the correct orientation and the transformed E. coli incubated as described above, ACC synthase activity was produced in the absence of IPTG at 20 nmol/h/mg protein and in presence of IPTG at 42 nmol/h/mg. ACC formation per 100 ml of culture was 2280 nmol without IPTG and 4070 nmol in the presence of IPTG. No ACC synthase activity or ACC production were observed when the 1-7 kb fragment was inserted in the opposite orientation.
A similar construct for tomato ACC synthase was used to test expression of the tomato cDNA in E. coli. The protein is synthesized as a fusion with a portion of the LacZ gene. The sequence at the junction is shown in Figure 15.
For construction of the vector containing this junction, pETC3C (Rosenberg, A.H., et al. Gene (1987) 56:125-155) was modified by cutting with EcoRI and ECoRV
and filling in with Klenow to remove a 375 by fragment downstream of the T7 promoter. The resulting religated plasmid was named pP07. pP07 was cut with BamHI and NdeI
and the large DNA segment was purified and ligated to a BaMHI/Ndel polylinker containing an EcoRI site to obtain the intermediate plasmid pP09.
The 1.4 kb EcoRI fragment from ptACCl was then ligated into the EcoRI site of pP09 to obtain the junction shown in Figure 15 and designated pP046.
This plasmid was then used to transform E. coli BL21 (DE3) (Rosenberg et al. (supra)). The cultures were induced by diluting fresh overnight cultures into 2x TY
(Maniatis et al. (supra) ) and grown at 37° to an absorp-tion at 600 nm of 0.7-0.8. IPTG was added to a final concentration of 2 mM and growth was continued for another WO 92/04456 2 Q 912 4 3 p~/US91 /06453 ,.-.,.

two hours. The cells were harvested and the recombinant polypeptide was purified as described by Nagai, K. and Thogersen, A.C., Meth Enzymol (1987) 153:461-481.
Figure 16 shows the synthesis of ACC synthase in nmol/15 ~1 of culture transformed with the tACC-containing vector in the presence and absence of IPTG. As shown in the figure, when the cDNA is ligated in the antisense direction, no ACC synthase is produced either in the presence or absence of IPTG (solid squares). When the cDNA is oriented in the correct orientation, after 180 min, over 2 nmol ACC synthase are produced after 15 ~1 in the presence of IPTG (solid circles), and between 0.5 and 1.0 nmol. in the absence of IPTG (open circles).
The production of ACC using labeled C'4-carboxyl labeled S-adenosyl-methionine is shown in Figure 17. In these figures, #1 is methionine, #2 is methionyl sulfite, #3 is methionyl sulfoxide, and #4 is unidentified. ACC is clearly labeled. Figure 17A shows the results in the absence of IPTG; a little ACC is formed. Figure 17C shows the results when the cDNA is ligated in the wrong orientation; no ACC is formed. Figure 17B shows the production of labeled ACC when the correct orientation of the cDNA is used. A large quantity of ACC is produced.
Example 7 Expression of Zucchini ACC Synthase in Yeast The EcoRI fragment representing cDNA clone ACC1 was subcloned into the EcoRI site of the yeast expression vector pBM258 (Johnston, M., et al. Mol Cell Biol (1984) 4_:1440-1448) and introduced into yeast strain YM2061. The yeast cells were grown on YP medium (Sherman, F., et al., Methods in Yeast Genetics (1979) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) at 37°C for 24 hr.
The medium either contained 2% galactose or 2% glucose.
After this culture, the cells were harvested and the supernatant was assayed for ACC released into the medium.
The pelleted cells were resuspended in buffer containing 5 gm glass beads and vortex-mixed 10 times for 30 sec and centrifuged at 2000 x g for 3 min. This supernatant was -..
-also collected. Solid ammonium sulfate was added to achieve 80% saturation an the precipitate was collected and dissolved in 2 ml of 20 mM Tris-HC1, pH 8.0, 10 ~,M
pyridoxal phosphate, 10 mM EDTA, 0.5 mM dithiothreitol;
and dialyzed against the same buffer. No ACC was produced in medium containing 2% glucose regardless of the construction of the vector. Control host vector and control vector with the ACC1 cDNA inserted in the anti-sense direction also gave no production of ACC in the cellular extract. However, when the medium contained 2%
galactose, the pBM-ACC1 vectors containing the cDNA in the correct orientation did show the production of ACC in the crude extract as well as ACC activity in the extracted protein. ACC synthase activity was 2.6 nmol/hr/mg protein in the crude extract; 354 nmol of ACC were formed per 100 ml of culture.
Example 8 Antisense Inhibition of Ethylene Production in Tomato Plants The ripening of tomatoes was shown to be preventable by the transformation of tomato plants with an antisense construction of the tomato ACC synthase gene which, therefore, putatively inhibited the synthesis of indige-nous ACC synthase. The cDNA clone was inserted in the opposite sense direction under the control of the cauli-flower CAMV 35S promoter and used to transform tomato plantlets using the A. tumefaciens mediated method. The regenerated plants produced tomatoes which failed to ripen, and which produced no ethylene at the times after pollination wherein ethylene was produced in control plants.
The antisense vector was constructed as follows: the 35S promoter was obtained as a 302 by fragment from pJ024D
(Ow, D.W., Proc Natl Acad Sci USA (1987) 84:4870-4874).
The plasmid pJ024D was digested with HindIII, treated with Klenow, and then cut with BamHI to isolate the 302 by fragment using gel electrophoresis. This was ligated to 3.5 kb of tomato ACC synthase cDNA by excising the coding WO 92/04456 2 0 912 ~3 p~/US91/06453 sequence from ptACCi by digestion with XbaI, filling with Klenow, and then cutting with BamHI. The two BamHI frag ments were ligated and the resulting ligation transformed into E. coli stain DHSA for cloning. The recovered plasmid was named pP032.
The plasmid pP032 was partially digested with SacI
and SalI and the digest was ligated into SalI/SacI
digested pBI101 binary Ti vector (Clonetech). pBI101 further contains the NOS 3' terminating sequences, as shown in Figure 17. The resultant vector, designated pP035 was transformed into E. coli DHSA for cloning. The sequences at the junctions were verified by sequence analysis.
pP035 or a control vector without the ACC-synthase gene was purified and introduced into Agrobacterium strain LBA4404 by a standard procedure described briefly as follows: A. tumefaciens LBA-4404 (2 ml) was grown over night at 28°C in LB broth, and this used to inoculate 50 ml of LB broth to obtain the desired culture. The inocu lated medium was grown at 28°C until the ODD was 0.5 -1Ø The cells were collected by centrifugation and the pellet was resuspended in 1 ml, 20 mM ice cold CaCl2. To 100 ~1 of the cell suspension, 1 ~m of pP035 DNA was added, and the mixture was incubated on ice for 30 min before snap-freezing in liquid nitrogen. The cells were then thawed at 37°C for 5 min and used to inoculate 1 ml LB. After 2 h growth at 28° C with agitation , 100 ~1 of the culture were plated on LB+Kanso medium; colonies appeared in 2-3 days at 28°C. The cells were recultured by picking several colonies and streaking on LB+Kan~
medium; again, 3-4 colonies were picked from independent streaks and 5 ml cultures in LB+KanSO medium were grown.
Stationary phase of these cultures were used for transfection of tomato plants.
The cells can be frozen using 15% glycerol at -80°C
to store for later use.
Preparation of Host Plants Tomato seeds were sterilized using a protocol which WO 92/ ~ ~ ~ ~ ~~ PCT/US91 /06453 consisted of treatment with 70% ethanol for 2 min with mixing; followed by treatment with 10% sodium hypochlorite and 0.1% SDS for 10 min with mixing, followed by treatment with 1% sodium hypochlorite, 0.1% SDS for 30 min with mixing, and washing with sterile water 3X for 2 min each wash.
For germination, 0.8 g of the sterilized seeds were placed in a Seed Germination Medium in a filled magenta box and grown for 2 weeks at low light in a growth room.
The magenta box contained 30 ml of the medium'.
After two weeks, when the seeds had germinated, cotyledons were dissected for the seedlings by cutting off the cotyledon tips and then cutting off the stem. This process was conducted in a large petri dish containing 5-10 ml of MSO.2 The harvested cotyledons were placed abaxial side up in tobacco feeder plates and grown for 48 h.
The feeder plates were prepared from a tobacco cell suspension in liquid medium3 at 25°C prepared with shaking at 130-150 rpm. The suspension was transferred to fresh medium at 1:10 dilution per every 3-5 days. 1 ml of rapidly dividing culture was placed on the feeder plate, overlaid with filter paper and placed in low light in a growth room. The feeder plates were supplemented with 10 ' Seed Germination Medium contains, per liter, 2.16 g of Murashige-Skoog salts; 2 ml of 500X B5 vitamins which had been stored at 20°C, 30 g sucrose and 980 ml water, brought to 1 N KOH and containing 8 g agar. The medium is autoclaved in 500 ml portions before filling the magenta boxes.
MSO contains per liter 4.3 g of Murashige-Skoog salts, 2 ml of 500 X B5 vitamins; 30 g of sucrose and 980 ml of water made 1 N in KOH to a final pH of 5.8.
Tobacco Suspension Medium contains in 1 liter 4.3 g Murashige-Skoog salts, 2 ml of 500X B5 vitamins, 30 g 3% sucrose, 10 ul of a 0.5 mg/ml solution of kinetin stored at -20°C, 2 ml of a 2 mg/ml solution of pCPA, and 980 ml of water made 1 N in KOH for a pH of 5.8 and autoclaved in 50 ml portions per 250 ml flask.

,,.-, ml Feeder Medium.4 The Agrobacterium containing the pP035 vector was inoculated into 50 ml LB containing kanamycin with a single colony of the strain. The culture was grown shaking vigorously at 30°C to saturation (OD>2.0 at 600 nm). The strain was chosen to come to full growth in less than 24 h. The culture was then diluted to 5 x l0a cell/ml with MSO nd split into 50 ml portions in plastic tubes.
Cotyledons from two of the feeder plates were scraped into each tube and rocked gently for 10-30 min. The cotyledons were then removed from the bacterial culture into sterile filter paper abaxial side up on a tobacco feeder plate and incubated for 48 h in low light in a growth room.
The cotyledons were then transferred axial side up to Callus Inducing Mediums In the Callus Inducing Medium, approximately four plates will be used per magenta box, and the explant are crowded. The box is place in a growth room for three weeks, and small masses of callus formed at the surface of the cotyledons. The explants are transferred to fresh plates containing the callus inducing medium every three weeks.
When the calli exceeded 2 ml, they were transferred 4 Feeder Medium contains 0.43 g Murashige-Skoog salts, 2 ml 500X B5 vitamins, 30 g of sucrose and 980 ml water made 1 N in KOH to a pH of 5.8, including 0.8% agar.
The foregoing components are autoclaved in two 500 ml portions and hormones are added when pouring plates to obtain 1 ~c/ml benzyl adenine (BA) and 0.2 ~g/ml of indole acetic acid (IAA).
Callus Inducing Medium contains per liter 4.3 g of Murashige-Skoog salts, 2 ml of 50oX B5 vitamins, 30 g of sucrose and 980 ml of water brought to 1 N KOH at a pH
of 5.8. The medium contains 0.8% agar and is autoclaved in two 500 ml portions. When poured into the plates, the following hormones are added to the following concentra-tions: l~Cm/ml BA, 0.2 ~,g/ml IAA, 100 ~g/ml kanamycin, 500 ~g/ml carbenicillin (Geopen).

-..,, 209~2~3 -to plates containing shoot inducing medium.b When the stem structure is evident, the shoots were dissected from the calli and the shoots were transferred to root inducing medium-containing plates.' After a vigorous root system was formed on the plants, the plantlets were transferred to soil. To do this, they were taken from the plates, removing as much agar as possible and placed in a high peat content soil in a small peat pot which fits into a magenta box with cover. When the seedling leaves reached the top of the box, the lid was loosened and continued to be uncovered slowly over a period of 4-5 days. The plants were then transferred to a light cart and larger pots, and kept moist.
The regenerated tomato plants were allowed to flower and pollinated. Seeds from the regenerated plants were replanted and grown to maturity. Flowers of these first generation plants were pollinated and tomatoes were developed and ethylene measured by gas chromatography at specified days after pollination. Figures 19 and 20 show the results for two sets of individual plants VI.1-4 (which contains the control vector) and AII.2-24 (which contains the antisense vector) in Figure 19 and VI.1-6 (which contains the control vector) and AII.2-7 (which contains the antisense vector) in Figure 20. As shown in these figures, in both cases, the control plants which had Shoot Inducing Medium contains, per liter, 4.3 g of Murashige-Skoog salts, 2 ml of 500X B5 vitamins, 0.6 g of MES and 900 ml of water made 1 N in KOH for a pH of 5.8, and 0.8% agar. The medium is autoclaved in two 450 ml portions and then is added 100 ml of a 30% filtered, sterilized glucose solution. When the plates are poured, additional components are added as follows: 0.1 mg/ml zeatin, 100 ug/ml kanamycin, 500 ~g/ml carbenicillin.
' Root Inducing Medium contains, per liter, 4.3 g Murashige-Skoog salts, 2m1 50oX B5 vitamins, 30 g of sucrose and 980 ml of water, 1 N in KOH to a pH of 5.8 in 0.8% agar. The medium is autoclaved in two 500 ml por-tions and when pouring plates, hormones are added to a concentration of 100 ~Cg/ml kanamycin and 500 ~Cg/ml or carbenicillin.

been transformed with control vector produced high levels of ethylene up to 8 ng/g fruit/h after approximately 50 days after pollination either in the presence of propylene or in the presence of air. However, in both cases, there was no production of ethylene in those plants which had been transformed with the antisense pP035 vector. In addition, the tomatoes which failed to produce ethylene also failed to ripen, whereas the control plants did ripen at this time.

Claims (9)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A DNR molecule comprising an expression system that is capable of generating, when contained in a plant host cell, a complementary RNA that is sufficiently complementary to an RNA transcribed from an endogenous ACC
synthase gene of tomato or zucchini to prevent synthesis of said endogenous ACC synthase, wherein said expression system comprises the reverse transcript of said complementary RNA, said reverse transcript being operably linked to control sequences that effect its transcription into said complementary RNA.

2. The DNA molecule of claim 1, wherein said ACC
synthase gene is either LE-ACC2 or pACC1.

3. A method for inhibiting ethylene production in a cell that normally produces ethylene, said method comprising, culturing in vitro or in vivo a cell that has been modified to contain the DNA molecule of any one of claims 1 or 2 under conditions wherein said complementary RNA is expressed.

4. The method of claim 3, wherein said cell is either a tomato cell or a zucchini cell.

5. The method of claim 3, wherein said complementary RNA complements RNA transcribed from either a tomato ACC
synthase gene or zucchini ACC synthase gene.

6. The plasmid designated as pP032.

7. The DNA molecule of claim 1, wherein said ACC
synthase gene comprises the nucleic acid sequence shown in Figure 1B.

8. The DNA molecule of claim 1, wherein said ACC
synthase gene comprises a nucleic acid selected from the group consisting of the nucleic acid sequences shown in Figures 8 and 13.

9. A DNA molecule comprising an expression system that is capable of generating, when contained in a plant host cell, a complementary RNA that is sufficiently complementary to an RNA transcribed from an endogenous ACC
synthase gene to prevent synthesis of said endogenous ACC
synthase, wherein said expression system comprises the reverse transcript of said complementary RNA, said reverse transcript being operably linked to control sequences that effect its transcription into said complementary RNA.